Previous studies have identified murine and human regulatory CD8+ T cells specific for TCR-Vβ families expressed on autologous activated CD4+ T cells. In the mouse, these regulatory CD8+ T cells were shown to be restricted by the MHC class Ib molecule, Qa-1. In the present study, we asked whether HLA-E, the human functional equivalent of Qa-1, binds Vβ peptides and whether the HLA-E/Vβ-peptide complex induces and restricts human CD8+ CTLs. We first created stable HLA-E gene transfectants of the C1R cell line (C1R-E). Two putative HLA-E binding nonapeptides identified in human TCR Vβ1 and Vβ2 chains (SLELGDSAL and LLLGPGSGL, respectively) were shown to bind to HLA-E. CD8+ T cells could be primed in vitro by C1R-E cells loaded with the Vβ1 (C1R-E/V1) or Vβ2 (C1R-E/V2) peptide to preferentially kill C1R-E cells loaded with the respective inducing Vβ peptide, compared with targets loaded with the other peptides. Priming CD8+ T cells with untreated C1R-E cells did not induce Vβ-specific CTLs. Of perhaps more physiological relevance was the finding that the CD8+ CTLs primed by C1R-E/V1 also preferentially killed activated autologous TCR Vβ1+. Similar results were observed in reciprocal experiments using C1R-E/V2 for priming. Furthermore, anti-CD8 and anti-MHC class I mAbs inhibited this Vβ-specific killing of C1R-E and CD4+ T cell targets. Taken together, the data provide evidence that certain TCR-Vβ peptides can be presented by HLA-E to further induce Vβ-specific CD8+ CTLs.